Exploration of universal cysteines in the binding sites of three opioid receptor subtypes by disulfide-bonding affinity labeling with chemically activated thiol-containing dynorphin A analogs

A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto grou p to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinit y labeling of mu and delta opioid receptors with SNpys-containing enkephali ns [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explo re the mercapto groups conserved at or near the ligand binding sites of thr ee opioid receptor subtypes, we synthesized two Cys(Npys)-containing analog s of dynorphin A, namely, [D-Ala(2),Cys(Npys)(8)] dynorphin A-(1-9) amide ( 1) and [D-Ala(2),Cys(Npys)(12)]dynorphin A-(1-13) amide (2). When rat (mu a nd delta) or guinea pig (kappa) brain membranes were incubated with these C ys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the n umber of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but al so label 6 receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled k appa receptors about 50 times more weakly than analog 1. These results sugg ested that dynorphin A analog 1 labels the Cys residues conserved in mu, de lta, and kappa receptors, whereas analog 2 only labels the Cys residues con served in mu and delta receptors.